Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1

Area of Science:

• Molecular Biology
• Biochemistry
• Virology

Background:

• DEAD-box helicases are crucial for RNA processing and cellular functions.
• Human DEAD-box protein 1 (DDX1) is implicated in tRNA/mRNA processing, cancer, and viral replication (e.g., HIV-1).
• The precise enzymatic mechanism of DDX1 remains largely uncharacterized.

Purpose of the Study:

• To elucidate the nucleotide and RNA binding mechanisms of human DDX1.
• To understand the kinetics and thermodynamics governing DDX1's enzymatic activity.
• To propose a model for DDX1's function based on binding properties.

Main Methods:

• Equilibrium titrations were used to determine binding affinities.
• Transient kinetics experiments were conducted to analyze reaction rates.
• Nucleotide and RNA binding interactions with DDX1 were quantitatively assessed.

Main Results:

• DDX1 exhibits exceptionally tight binding to adenosine diphosphate (ADP), with affinities three orders of magnitude stronger than for adenosine triphosphate (ATP).
• This tight ADP binding suggests DDX1 can become trapped in an inactive conformation, necessitating nucleotide exchange factors for its recycling.
• Cooperative binding of RNA and ATP to DDX1 was observed, impacting ATP hydrolysis rates.

Conclusions:

• DDX1's enzymatic cycle likely involves nucleotide exchange factors to release tightly bound ADP.
• The observed cooperativity in RNA and ATP binding influences DDX1's catalytic activity.
• A conformational model is proposed where ATP or RNA binding alone induces a partial shift to a 'closed' state, with limited further change upon binding both.